This Exploratory/Developmental Research (R21) application proposes to develop an optical-phase- conjugation-assisted (OPC) 4Pi microscope that is capable of high lateral (200 nm) and axial (120 nm) resolution imaging. A typical 4Pi microscope consists of two counter-propagating laser beams that are focused to the same spatial point. The mutual interference of the two beams allows an improvement in the axial resolution by a factor of 5-7 versus a standard confocal microscope. However, a 4Pi microscope performs poorly for optically inhomogeneous samples as extensive scattering degrades the focal spot quality and can lead to a misalignment of the two focal spots. Furthermore, 4Pi microscopes are generally difficult to implement and use well because the exact superposition of the laser beams'focal spots is critical but easily disrupted. Our proposed research will leverage our current expertise in optical phase conjugation (OPC), time-reversal turbidity suppression via OPC, and tissue optics to significantly improve the capability of 4Pi microscopy. Optical phase conjugation can be roughly interpreted as the time-reversed playback of a target light field. We have recently established that, because scattering is deterministic, it is possible undo tissue scattering by time- reversing a scattered light field through the target tissue. We aim to demonstrate that the incorporation of a specially adapted OPC arrangement in a 4Pi microscope will allow such a microscope to adaptively align its focus and to correct sample aberration which has been an obstacle for the conventional 4Pi system. The application range of the proposed OPC 4Pi microscope is potentially broad. The anticipated aberration compensation ability will allow this system to image thicker and more heterogeneous samples. We expect that such systems will be useful in neuroscience and developmental biology experiments where precise 3D mapping of complex samples, such as embryos, are important. PUBLIC HEALTH RELEVANCE: The successful completion of this proposed project will result in more robust 4Pi microscope systems that are simpler to use and capable of imaging through thicker and more heterogeneous samples. We expect that such systems will be useful in neuroscience and developmental biology experiments where precise 3D mapping of complex samples, such as embryos, are important. The proposed work will also further our current research effort on the use of time-reversed light field to induce tissue transparency - a research direction that can ultimately lead to accurate and non-invasive biochemical sensing, and light-based deep tissue imaging.